微泡
四斯潘宁
内体
外体
细胞生物学
内吞循环
全内反射荧光显微镜
活体细胞成像
分泌物
胞吐
脂质双层融合
内吞作用
小泡
CD63
化学
荧光显微镜
细胞外
生物
细胞
膜
细胞内
生物化学
荧光
小RNA
物理
基因
量子力学
作者
Matthew F. Pescosolido,Qing Ouyang,Judy S. Liu,Eric M. Morrow
出处
期刊:Methods in molecular biology
日期:2023-01-01
卷期号:: 213-220
被引量:1
标识
DOI:10.1007/978-1-0716-3287-1_17
摘要
Exosomes represent a class of extracellular vesicles (EVs) derived from the endocytic pathway that is important for cell-cell communication and implicated in the spread of pathogenic protein aggregates associated with neurological diseases. Exosomes are released extracellularly when multivesicular bodies (also known as late endosomes) fuse with the plasma membrane (PM). An important breakthrough in exosome research is the ability to capture MVB-PM fusion and exosome release simultaneously in individual cells using live-imaging microscopy techniques. Specifically, researchers have created a construct fusing CD63, a tetraspanin enriched in exosomes, with the pH-sensitive reporter pHluorin whereby CD63-pHluorin fluorescence is quenched in the acidic MVB lumen and only fluoresces when released into the less acidic extracellular environment. Here, we describe a method using this CD63-pHluorin construct to visualize MVB-PM fusion/exosome secretion in primary neurons using total internal reflection fluorescence (TIRF) microscopy.
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